Notch receptors anchor a fundamental signaling pathway conserved from sea urchins to humans. Signals transduced by these receptors normally influence cell fate decisions during development, and regulate cell growth, differentiation, and death in a variety of tissue types. Aberrant Notch signals have also been implicated in vascular disease, neurodegeneration, and cancer. Notch receptors normally reside on the cell surface in a resting conformation, which is maintained by a negative regulatory region (NRR) that encompasses three Lin12/Notch (LNR) repeats and a heterodimerization (HD) domain immediately external to the membrane. Ligand binding induces proteolytic sensitivity at a metalloprotease cleavage site within the HD domain, with proteolysis at this site triggering subsequent cleavage by gamma-secretase to release the intracellular portion of Notch (ICN) from the membrane. ICN then enters the nucleus and induces transcription of target genes by driving the assembly a Notch transcriptional activation complex (NTC) that includes a DNA-bound transcription factor called CSL and a co-activator protein of the Mastermind-like (MAML) family. The overarching goal of these studies is to elucidate the molecular logic of Notch signaling in different developmental, physiologic, and pathophysiologic contexts. Our studies will focus on two key steps in the activation of Notch signals: how is metalloprotease cleavage prevented prior to ligand binding, and how do the nuclear complexes assemble to induce transcription of target genes? In the next period of support, we will pursue the following specific aims: (i) elucidate structural and mechanistic features of the negative regulatory region underlying Notch autoinhibition and activation, and (ii) determine the structure of an NTC dimer on the paired CSL binding site from the HES-1 promoter and investigate implications of dimerization on transcription of different Notch target genes. Our findings will lead to new insights into the mechanism of Notch signaling, and will have broad relevance to the pathogenesis of vascular disease, neurodegeneration, and cancer. PUBLIC HEALTH RELEVANCE: Notch receptors are cell-surface proteins that communicate signals required for proper development of organisms ranging from sea urchins to man, yet mutations of Notch genes can lead to human developmental disorders, and aberrant Notch signaling contributes to the development of a number of other diseases, including cancer. To carry out their primary function as developmental regulators, Notch receptors are converted from a resting state to an active one, in which the protein acquires sensitivity to a processing step allowing the active receptors to form molecular partnerships with themselves and with other proteins to switch on the so called transcription machinery that makes the products of other genes. In the studies proposed here, we plan to determine (i) how the receptors are converted from resting to active in normal cells and in leukemic cells that have activating mutations of the receptor, and (ii) how the self- partnerships of Notch receptors are formed, and how they influence Notch function in normal cells and in cancer cell lines; our findings will thus inform efforts to develop mechanism-based modulators of Notch signaling as potential therapeutics for cancer and other diseases.